1. Volume 87, Issue 3, Pages 531-542 (November 1996)
Uncoupling of Grb2 from the Met Receptor In Vivo Reveals Complex Roles in Muscle Development 
Flavio Maina, Franca Casagranda, Enrica Audero, Antonio Simeone, Paolo M Comoglio, Rüdiger Klein, Carola Ponzetto 
Cell 
Volume 87, Issue 3, Pages (November 1996)
DOI: /S (00)

2. Figure 1 Knock-In of Point Mutations in the met Locus
(A) Diagram of the targeting vector, wild-type and mutant alleles, and resulting chimeric protein. The human MET cDNA fragment fused in-frame with the third exon of the genomic clone, codes for the transmembrane and cytoplasmic domain of the Met receptor. After homologous recombination, the neo cassette remains within the mutant allele. N indicates cleavage sites for NdeI. The 5′ probe identifies a 16 kb NdeI DNA fragment in the wild-type allele and a 12 kb NdeI DNA fragment in the mutant allele (see [B]). Notations “1” and “2” indicate the location of the oligonucleotides used for PCR screening of the double-selected ES cells. The chimeric protein can specifically be recognized by anti-human Met antibodies raised against the unique carboxy-terminal peptide. Asterisk indicates the carboxy-terminal SH2 multifunctional docking sites (amino acids are numbered according to the human c-MET DNA sequence deposited at EMBL/GenBank with accession number X54559). MetWT: chimeric protein with wild-type multifunctional tyrosines. MetGrb2: chimeric protein with disrupted Grb2 binding site. MetD: chimeric protein with mutated multifunctional tyrosines.
(B) Southern blots of NdeI digests of genomic DNA isolated from double-selected ES cell clones (lanes 1–11), transfected with the metWT, metD, and metGrb2 constructs. Lane 2: ES cell clone in which both alleles were targeted. Lanes R: R1 ES cell control DNA. The probe used (position shown in [A]) identifies a 16 kb NdeI DNA fragment in the wild-type allele and a 12 kb NdeI fragment in the recombinant allele.
(C) Northern blot of total RNA isolated from liver of wild-type (+/+), metWT/+ (+/−) and metWT/WT (−/−) mice. The higher 8.8 kb band is the endogenous transcript, and the 7.2 kb band is the recombinant transcript. The difference in size is due to the fact that in the recombinant allele, the 3′ untranslated region is substituted by the 1.2 kb β-globin 3′ untranslated region and polyadenylation site. The probe used was a mouse met cDNA fragment located in the extracellular domain (see Experimental Procedures) common to both alleles.
(D) Immunoprecipitation and Western blot of extracts of E15.5 homozygous and control (+/+) embryos. Left: the antibody used for immunoprecipitation and Western blotting was specific for the human Met protein. Right: the antibody used for immunoprecipitation and Western blotting was specific for the mouse Met protein.
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3. Figure 2
Loss of the Met Multifunctional Docking Sites Results in Lack of Muscles Deriving from Migratory Myoblasts
Hematoxylin–eosin staining of sections of diaphragm (A–C) and myosin staining of sections of forelimbs (D–F), back (G–I), and rib cage (J–L) of E15.5 embryos of the indicated genotypes. In metD/D mutant embryos, diaphragm and forelimb muscles are absent, while back and intercostal muscles are normal. lu, lung; d, diaphragm; li, liver; h, homerus; r, rib. Magnification: 400 × (A–C); 100 × (D–I), 200 × (J–L).
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4. Figure 3
Uncoupling Grb2 from Met Is Compatible with Term Development and Affects Muscles Deriving from Migratory Precursors
(A–C) Homozygous mutant mice of the indicated genotype at P0. Forelimbs of metGrb2/Grb2 mice are hyperflexed. metGrb2/D mice are born alive, but they retain a fetal position and die a few minutes after birth.
(D–I) Sections of the forearm of mutant mice at P0, taken halfway between the wrist and the elbow (D–F) and at the level of the elbow (G–I). Hematoxylin–eosin staining shows a graded reduction in muscle mass, progressively more severe in metGrb2/Grb2 and metGrb2/D mutants.
(J–L) Sections of the diaphragm stained with phalloidin. u, ulna; r, radius; lu, lung; d, diaphragm; li, liver. Magnification: 50 × (D–I); 400 × (J–L).
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5. Figure 4
Loss of the Link with Grb2 Does Not Affect Differentiation of Embryonal Myoblasts or Their Migration out of the Somites
(A–C) Whole-mount hybridization of E11.5 embryos with a MyoD probe. Myogenic cells in the axial and neck muscles (wide arrow) are unaffected by the Met mutations. Developing appendicular muscles (thin arrow) are reduced in metGrb2/Grb2 embryos, but they are positive for expression of MyoD. metD/D embryos lack myogenic cells in shoulders and limbs.
(D–F) Whole-mount hybridization of E10 embryos with a met probe. Myoblast precursors in metWT/WT and metGrb2/Grb2 embryos leave the somite and enter the limb bud, while they do not migrate out of the somite in metD/D embryos. Probes are described in Experimental Procedures.
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6. Figure 5
Loss of the Link with Grb2 Affects Development of Muscles That Do Not Originate from Migratory Precursors
Phalloidin staining of middle tongue (A–C) and intercostal muscles (D–I) of mutant mice at P0. The muscle structure appears to be progressively more disorganized in metGrb2/Grb2 and metGrb2/D mutants. Fibers are diminished in number and seem to be dissociated from each other. Close-up of hematoxylin–eosin staining of back muscles (J–L) shows empty spaces between fibers and a reduction in fiber caliber. ap, apical surface of the tongue; b, body wall muscles; r, rib. Magnification: 100 × (A–F), 200 × (G–I), 400 × (J–L).
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7. Figure 6
Loss of the Link with Grb2 Affects Development of Secondary Fibers
(A–C) Representative sections of intercostal muscles at E17.5 double-stained with anti-myosin (MAb MF20 and rhodamin-conjugated goat anti-mouse) and anti-laminin (polyclonal L9393 and fluorescein-conjugated goat anti-rabbit), used to count secondary fibers (marked with asterisks).
(D–F) BrdU-labeled nuclei in sections of intercostal muscle of E17.5 embryos. Pregnant mothers were injected 2 days earlier. Counts of labeled nuclei were made on BrdU/laminin overlays (insert; laminin was detected using rhodamin-conjugated goat anti-rabbit sera). Only nuclei found underneath basal laminas (arrow) were counted. Arrowheads show examples of nuclei outside fibers. Magnification: 630 × (A–C); 400 × (D–F).
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8. Figure 7 Met and Grb2 Are Coexpressed in Fetal Myoblasts
(A) In situ hybridization of intercostal (left) and back (right) muscles of E15.5 embryos with a met-specific probe (described in Experimental Procedures). r, ribs. Magnification: 100 ×.
(B) Western blots of immunoprecipitates obtained from extracts (equalized for the amount of total proteins) of primary cultures of myogenic cells derived from E16.5 embryos. Lanes 1–4: Met immunoprecipitates of liver (lane 1), myoblasts (lane 2), myotubes (lane 3), and fibroblasts (lane 4). Lanes 5–9: Grb2 immunoprecipitates of C2C12 myogenic cells undifferentiated (lane 5) and differentiated (lane 6), myoblasts (lane 7), myotubes (lane 8), and fibroblasts obtained from the preplating step (lane 9; see Experimental Procedures).
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